The present invention relates to novel protein tyrosine phosphatase modulating compounds, to methods for their preparation, to compositions comprising the compounds, to their use for treatment of human and animal disorders, to their use for purification of proteins or glycoproteins, and to their use in diagnosis. The invention relates to modulation of the activity of molecules with phosphotyrosine recognition units, including protein tyrosine phosphatases (PTPases) and proteins with Src-homology-2 domains, in in vitro systems, microorganisms, eukaryoic cells, whole animals and human beings.
Reversible phosphorylation of proteins is a prevalent biological mechanism for modulation of enzymatic activity in living organisms. Tonks et al., J. Biol. Chem., 263(14):6722-30 (1988). Such reversible phosphorylation requires both a protein kinase (PK), to phosphorylate a protein at a particular amino acid residue, and a protein phosphatase (PP), to remove the phosphate moieties. See generally, Hunter, Cell, 80:225-236 (1995). Recently, it has been estimated that humans have as many as 2000 conventional PK genes, and as many as 1000 PP genes. Id.
One major class of PK""s/PP""sxe2x80x94the protein seine/threonine kinases and protein serine/threonine phosphatasesxe2x80x94have been shown to play critical roles in the regulation of metabolism. See generally, Cohen, Trends Biochem. Sci., 17:408-413 (1992); Shenolikar, Ann. Rev. Cell Biol., 10:55-86 (1994); Bollen et al., Crit. Rev. Biochem. Mol. Biol., 27:227-81 (1992). As their name suggests, these enzymes phosphorylate and dephoshphorylale serine or threonine residues of substrate proteins. Inhibitors of prntein serine/threonine phosphatases and kinases have been described. See, e.g., MacKintosh and MacKintosh, TIBS, 19:444-448 (1994).
The protein tyrosine kinases/phosphatases comprise a second, distinct family of PK/PP enzymes of significant interest, and have been implicated in the control of normal and neoplastic cell growth and proliferation. See Fisher et al., Science, 253:401-406 (1991) Protein tyrosine kinase (PTK) genes are ancient in evolutionary origin and share a high degree of inter-species conservation. See generally Hunter and Cooper, Ann. Rev. Biochem., 54:897-930 (1985). PTK enzymes exhibit high specificity for tyrosine, and ordinarily do not phosphorylate serine, threonine, or hydroxyproline.
More than 75 members of the PTPase family have been identified in eukaryotes, prokaryotes, and even viruses. Tonks and Neel, Cell 87:365-368. Protein tyrosine phosphatases (PTPases) were originally identified and purified from cell and tissue lysates using a variety of artificial substrates, and therefore their natural functions and substrates were not obvious. However, their roles in cellular processes including cell-cell contact and cell adhesion, and growth factor and antigen signaling events, have begun to be elucidated.
PTPases are generally grouped into two categories: those which have both an extracellular domain and an intracellular catalytic domain, the receptor PTPases (R-PTPases); and those which are entirely intracellular. For R-PTPases much effort has been directed at determining the function of the extracellular domain. Most of the R-PTPases contain extracellular domains which are structurally similar to domains found in known adhesion molecules; these domains include fibronectin type III repeats, immunoglobulin domains, and cadherin extracellular repeats. See generally Brady-Kalnay and Tonks, Curr. Opin. Cell. Biol. 7:650-657 (1995); Streuli, Curr. Opin. Cell. Biol. 8:182-188 (1996). This homology with proteins known to be involved in adhesion suggested a role for these R-PTPase in regulating or mediating adhesion events. For several of the R-PTPases, this has now been demonstrated.
Cells form specialized structures at the sites of cell-cell contact (adherens junctions) and cell-extracellular matrix contact (focal adhesion). Multiple signal transduction molecules are recruited to these sites, including several PTK""s; and these sites are characterized by increased protein tyrosine phosphorylation. These sites are impermanent, and are created and destroyed as required for cell mobility. As enhanced tyrosine phosphorylation is characteristic of the formation of adherens junctions and focal adhesions, it is likely that protein tyrosine dephosphorylation by PTPases serves to regulate the creation and destruction of the sites. Supporting this, several studies have shown that treatment with a general PTPase inhibitor (vanadate) resulted in increased focal adhesion formation and increased cell spreading. Volberg et al., The EMBO J. 11:1733-1742 (1992); Bennett al,. J. Cell Sci. 106:891-901 (1993). Importantly, the broadly-expressed LAR R-PTPase has been demonstrated to localize to focal adhesions, apparently via the LAR-interacting protein LIP.1. Serra-Pages et al., The EMBO J. 14:2827-2838 (1995). As PTPxcex4 and PTP"sgr", both R-PTPases, also associate with LIP.1 [Pulido et al., Proc. Natl. Acad. Sci. 92:11686-11690 (1995)], it is likely that these two phosphatases can also localize to focal adhesions. Most significantly, LAR only localized to the portion of the focal adhesion which is proximal to the nucleus, and is thought to be undergoing disassembly. Thus it is likely that these phosphatases act to negatively regulate focal adhesion formation, acting to enhance the destruction of the focal adhesion site.
R-PTPases may also act to positively regulate adhesion. Adherens junctions contain, among others, adhesion receptors termed cadherins which mediate cell-cell contact through homophilic binding; the cadherins associate with xcex1-, xcex2-, and xcex3-catenins, intracellular proteins which interact with cortical actin. Association between cadherins and catenins serves to stabilize the adherens junction and to strengthen cell-cell contact. See generally Cowin, Proc. Natl. Acad. Sci. 91:10759-10761 (1994). Association of cadherin with xcex2-catenin is decreased by tyrosine phosphorylation of xcex2-catenin [Kinch el al., J. Cell. Biol. 130:461-471 (1995); Behrens et al., J. Cell. Biol. 120:757-766 (1993)]; moreover, treatment with the PTPase inhibitor vanadate inhibits cadherin-dependent adhesion [Matsuyoshi et al., J. Cell. Biol. 118:703-714 (1992)]. Collectively, these data indicate that PTPase activity is critical in maintaining cadherin-mediated cell aggregation. The R-PTPases PTPxcexc and PTPxcexa associate intracellularly with cadherins, and colocalize with cadherins and catenins to adherens junctions [Brady-Kalnay et al., J. Cell. Biol. 130:977-986 (1995); Fuchs et al., J. Biol. Chem. 271:16712-16719 (1996)], thus PTPxcexc and PITxcexa are likely to enhance cadherin function by limiting catenin phosphorylation.
In addition to their catalytic function in regulating adhesion events, several R-PTPases have direct roles in mediating adhesion through their extracellular domains. PTPxcexa and PTPxcexc mediate cellular aggregation through homophilic binding [Brady-Kalnay et al., J. Cell. Biol. 122:961-972 (1993); Gebbink et al., J. Biol. Chem. 268:16101-16104 (1993); Sap et al,. Mol. Cell. Biol. 14:1-9 (1994)]. The neuronal PTPxcex6 (which has also been called R-PTPxcex2) binds to contactin, a neuronal cell recognition molecule; binding of PTPxcex6 to contactin increases cell adhesion and neuritc outgrowth. Peles et al., Cell 82:251-260 (1995). A secreted splice variant of PTPxcex6 (also known as phosphacan) binds the extracellular matrix protein tenascin [Barnea et al. J. Biol. Chem. 269:14349-14352 (1994)], and the neural cell adhesion molecules N-CAM and Ng-CAM [Maurel et al., Proc. Natl. Acad. Sci. 91:2512-2516 (1994)]. As the expression of PTPxcex6 is restricted to radial glial cells in the developing central nervous system, which are though to form barriers to neuronal migration during embryogenesis, it is likely that the interaction of PTPxcex6 with contactin, tenascin, N-CAM, and/or Ng-CAM acts to regulate neuronal migration. This has been demonstrated for a related R-PTPase, DLAR, in Drosophila [Krueger et al. Cell 84:611-622 (1996)].
Because tyrosine phosphorylation by PTK enzymes usually is associated with cell proliferation, cell transformation and cell differentiation, it was assumed that PTPases were also associated with these events. For several of the intracellular PTPases, this function has now been verified.
SHP1 (which has also been called SHPTP1, SHP, HCP, and PTP-1C [see Adachi et al., Cell 85:15 (1996)]), an intracellular PTPase which contains two amino-terminal phosphotyrosyl binding Src Homology 2 (SH2) domains followed by the catalytic PTPase domain, has been demonstrated to be an important negative regulator of growth factor signaling events. See generally Tonks and Neel, supra: Streuli, supra. In mice, loss of SHP1 function (the motheaten and viable motheaten phenotypes) causes multiple hematopoietic defects resulting in immunodeficiency and severe autoinmmunity; culminating in lethality by 2-3 weeks or 2-3 months depending on the severity of SHP1 deficiency. Although these mice have reduced numbers of hematopoietic cells, suggesting defects in development and maturation, those cells which survive and enter the periphery are characterized by hyper-responsiveness to growth factors and antigen. This observation suggested a role for SHP1 in negative regulation of hematopoietic signaling events.
This has now been well established for the erythropoietin receptor (EpoR), a member of the cytokine receptor family (which also includes the receptors for interleukins 2, 3, 4, 5, 6, 7; granulocyte-macrophage colony stimulating factor, and macrophage colony stimulating factor). SHP1 associates via its SH2 domains with tyrosine-phosphorylated EpoR, causing dephosphorylation and inactivation of the EpoR-associated Janus kinase 2 and termination of the cellular response to erythropoietin. Klingmuller et al., Cell 80:729-738 (1995). Mutation of the tyrosine on the EpoR to which SHP1 binds results in enhanced cell proliferation to erythropoietin in vitro [Klingmuller, supra]. In humans, mutation of the EpoR resulting in loss of association with SHP1 causes autosomal dominant benign erythrocytosis, which is characterized by increased numbers of erythrocytes in the periphery and increased hematocrit. de la Chapelle et al., Proc. Natl. Acad. Sci. 90:4495-4499 (1993).
SHP1 also appears to be a negative regulator of the cellular response to colony stimulating factor-1 (CSF-1, a major macrophage mitogenic cytokine), as cells from viable motheaten and motheaten mice, which have reduced or absent SHP1 function, are hyper-resporsive to CSF-1 in vitro. Reduced SHP1 expression also results in increased cellular response to interleukin 3 [Yi et al., Mol. Cell. Biol. 13:7577-7586 (1993)]. Collectively, these observations suggest that SHP1 functions to limit the a cellular response to cytokines and growth factors by reversing the tyrosine phosphorylation of key signaling intermediates in these pathways.
PTPases appear to play a homologous role in the insulin signaling pathway. Treatment of adipocytes with the PTPase inhibitor vanadate results in increased tyrosine phosphorylation and tyrosine kinase activity of the insulin receptor (InsR), and enhances or mimics the cellular effects of insulin including increased glucose transport. See, e.g., Shisheva and Shechter, Endocrinology 133:1562-1568 (1993); Fantus, et al., Biochemistry 28:8864-8871 (1989); Kadota, et al., Biochem. Biophys. Res. Comm. 147:259-266 (1987); Kadota, et al., J. Biol. Chem. 262:8252-8256 (1987). Transiently induced reduction in expression of two PTPases, the intracellular PTPase PTP-1B and the R-PTPase LAR, resulted in similar increases in the cellular response to insulin. Kulas, et al., J. Biol. Chem. 270:2435-2438 (1995); Ahmad et al., J. Biol. Chem. 270:20503-20508 (1995). Conversely, increased cellular expression of several PTPases (PTPxcex1, PTPxcex5, CD45) in vitro has been demonstrated to result in diminished InsR signaling [see. e.g., Moller, et al., J. Biol. Chem. 271:23126-23131 (1995); Kulas et al., J. Biol. Chem. 271:755-760 (1996)]. Finally, increased expression of LAR was observed in adipose tissue from obese human subjects [Ahmad, et al., J. Clin. Invest. 95:2806-2812 (1995)]. These data provide clear evidence that PTPases negatively regulate the insulin signaling pathway.
While many of the PTPases function to negatively regulate cellular metabolism and response, it is becoming increasingly evident that PTPases provide important positive signaling mechanisms as well. Perhaps the beat example of such a positive regulator is the hematopoietic R-PTPasc CD45. See generally Streuli, supra; Okumura and Thomas, supra; Trowbridge, Annu. Rev. Immunol. 12:85-116 (1994). CD45 is abundantly expressed on the cell surface of all nucleated hematopoietic cells. in several alternative splice variants. T and B lymphocytes which lack CD45 expression are incapable of responding normally to antigen, suggesting that CD45 is required for antigen receptor signaling. Genetically engineered mice which lack expression of CD45 exhibit severe defects in T lymphocyte development and maturation, indicating an additional role for CD45 in thymopoiesis. The major substrates for CD45 appear to be members of the Src family of PTK""s, particularly Lck and Fyn, whose kinase activity is both positively and negatively regulated by tyrosine phosphorylation. Lck and Fyn isolated from CD45-deficient cells are hyperphosphorylated on negative regulatory tyrosine residues, and their PTK activity is reduced. As CD45 can dephosphorylate and activate purified Lck and Fyn in vitro, these data suggest that CD45 maintains the activity of Lck and Fyn in vivo through dephosphorylation of these negative regulatory tyrosines and that this is an important mechanism for maintaining lymphocyte homeostasis.
A second PTPase which is now believed to play an important positive role in signal transduction is the intracellular. SH2-domain-containing SHP2 (which las also been called SHPTP-2, SHPTP-3, syp, PTP2c, and PTP-1D [Adachi, et al., supra]). See generally Saltiel, Am. J. Physiol. 270:E375-385 (1996); Draznin, Endocrinology 137:2647-2648. SHP2 associates, via its SH2 domains, with the receptor for platelet-derived growth factor (PDGF-R), the receptor for epidermal growth factor (EGF-R). with the insulin receptor, and with the predominant substrate of the lnsR. insulin receptor substrate 1 (IRS1). Bennett, et al., Proc. Natl. Acad .Sci. 91:7335-7339 (1994); Case, et al., J. Biol. Chem. 269:10467-10474 (1994); Kharitonenkov, et al., J. Biol. Chem. 270:29189-29193 (1995); Kuhne, et al., J. Biol. Chem. 268:11479-11481 (1993). SHP2 PTPase activity is required for cellular response to EGF and insulin as competitive expression of inactive forms of SHP2 results in diminished signaling events and reduced cellular responses to EGF and insulin. Milarski and Saltiel, J. Biol. Chem. 269:21239-21243 (1994); Xiao et al., J. Biol. Chem. 269:21244-21248 (1994); Yamauchi el al., Proc. Natl. Acad. Sci. 92:664-668 (1995). The relevant substrate(s) for the PTPase domain of SHP2 is not known.
Due to the fundamental role that PTPases play in normal and neoplastic cellular growth and proliferation. A need exists in the art for agents capable of modulating PTPase activity. On a fundamental level, such agents are useful for elucidating the precise role of protein tyrosine phosphatases and kinases in cellular signalling pathways and cellular growth and proliferation. See generally MacKintosh and MacKintosh, TIBS, 19:444-448 (1994).
More importantly, modulation of PTPase activity has important clinical significance. For example, PTP-1B overexpression has been correlated with breast and ovarian cancers [Weiner et al., J. Natl. Cancer Inst., 86:372-8 (1994); Weiner et al., Am J. Obstet. Gynecol., 170:1177-883 (1994)], and thus agents which modulate PTP-1B activity would be helpful in elucidating the role of PTP-1B in these conditions and for the development of effective therapeutics against these disease states. The important role of CD45 in hematopoietic development and T lymphocyte function likewise indicates a therapeutic utility for PTPase inhibitors in conditions that are associated with autoimmune disease, and as a prophylaxis for transplant rejection. The antibiotic suramin, which also appears to possess anti-neoplastic indications, has recently been shown to be a potent, irreversible, non-competitive inhibitor of CD45. See Ghosh and Miller, Biochem. Biophys. Res. Comm. 194:36-44 (1993). The negative regulatory effects of several PTPases on signaling through receptors for growth factors and cytokines, which are implicated in normal cell processing as well as discase states such as cancer and atherosclerosis, also indicate a therapeutic potential for PTPase inhibitors in diseases of hematopoietic origin.
The PTPase Yop2b is an essential virulence determinant in the pathogenic bacterium Yersinia, responsible for bubonic plague. Bliska et al., Proc. Natl. Acad. Sci. USA. 88:1187-91 (1991), and thus an antimicrobial indication exists for PTPase inhibitor compounds, as well.
PTPases have been implicated in diabetic conditions. The relevents with one family of PTPase inhibitors, vanadium derivatives, indicate a therapeutic utility for such compounds as oral adjuvants or as alternatives to insulin for the treatment of hyperglycemia, See Posner et al., J. Biol. Chem., 269:4596-4604 (1994). However, such metal-containing PTPase inhibitors act in a fairly nonspecific fashion and act with similar potencies against all PTPase enzymes.
In addition to vanadium derivatives, certain organic phosphotyrosine minmetics are reportedly capable of competitively inhibiting PTPase molecules when such mimetics are incorporated into polypeptide artificial PTPase substrates of 6.11 amino acid residues. For example, a xe2x80x9cnaturalxe2x80x9d (phosphorylated tytosine) PTPase substrate, which may be depicted by the Formula: 
has been mimicked by eleven-mer oligopeptides containing phosphonomethyl phenvlalanine (Pmp), as depicted by the schematic Formula: 
See Chatterjee et al., xe2x80x9cPhosphopeptide substrates and phosphonopeptide inhibitors of protein tyrosine phosphatases,xe2x80x9d in Pepindes: Chemistry and Biology (Rivier and Smith, Eds.), 1992, Escom Science Publishers; Leiden, Netherlands, pp. 553-55; Burke et al., Biochemistry, 33:6490-94 (1994). More recently, Burke et al., Biochem. Biophys. Res. Comm. 204(1):129-134 (1994) reported that a particular hexameric peptide sequence comprising a Pmp moiety or, more preferably, a phosphonodifluoromethyl phenylalanine (F2Pmp) moiety, as depicted by the schematic Formula: 
competitively inhibited PTP-1B. However, such hexapeptide inhibitors nonetheless possess drawbacks for PTPase modulation in vivo. More particularly, the hexapeptide inhibitors described by Burke et al. are sufficiently large and anionic to potentially inhibit efficient migration across cell membranes, for interaction with the catalytic domains of transmembrane and intracellular PTPase enzymes which lie within a cell membrane. A need exists for small, organic-molecule based PTPase inhibitors having fewer anionic moieties to facilitate migration across cell membranes.
For all of the foregoing reasons, a need exists in the art for novel compounds effective for modulating, and especially inhibiting, the phosphatase activity of protein tyrosine phosphatase molecules.
The invention provides compounds and derivatives thereof useful for modulating, wad especially inhibiting, the phospbatase activity of one or more protein tyrosine phosphatase (PTPase) and/or dual specificity phosphatase enzymes. In one aspect, the present invention relates to compounds having the general structure shown in Formula (A1): 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3, X and Y are defined below. The inventions further provides salts, esters, prodrugs, solvates, and the like of the compounds, and compositions comprising these compounds.
In the specification and claims the term xe2x80x9cderivativesxe2x80x9d means: aryl acrylic acids with structure depicted in Formula (A1) having substitution (with, e.g., hydrogen, hydroxy, halo, amino, carboxy, nitro, cyano, methoxy, etc.) at one or more atoms of the aryl ring. Moreover, xe2x80x9cderivativesxe2x80x9d includes compounds of the Formula (A1) having substitution at the alkene carbons with, e.g., an electron withdrawing group (e.g., Cl, F, Br, CF3, phenyl) or an electron donating group (e.g., CH3, alkoxy). 
As used herein, the term xe2x80x9cattachedxe2x80x9d signifies a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art.
The terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d include fluorine, chlorine, bromine, and iodine.
The term xe2x80x9calkylxe2x80x9d includes C1-C11 straight chain saturated and C2-C11 unsaturated aliphatic hydrocarbon groups, C1-C11 branched saturated and C2-C11 unsaturated aliphatic hydrocarbon groups, C3-C8 cyclic saturated and C5-C8 unsaturated aliphatic hydrocarbon groups, and C1-C11 straight chain or branched saturated and C2-C11 straight chain or branched unsaturated aliphatic hydrocarbon groups substituted with C3-C8 cyclic saturated and unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, this definition shall include but is not limited to methyl (Me), ethyl (Et), propyl (Pr), butyl (Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, isopropyl (i-Pr), isobutyl (i-Bu), tert-butyl (t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, methylcyclopropyl, ethylcyclohexenyl, butenylcyclopentyl, and the like.
The term xe2x80x9csubstituted alkylxe2x80x9d represents an alkyl group as defined above wherein the substitutents are independently selected from halo, cyano, nitro, trihalomethyl, carbamoyl, C0-11alkyloxy, arylC0-11alkyloxy, C0-11alkylthio, arylC0-11alkylthio, C0-11alkylamino, arylC0-11alkylamino, di(aryC0-11alkyl)amino, C1-11alkylcarbonyl, arylC1-11alkylcarbonyl, C1-11alkylcarboxy, arylC1-11alkylcarboxy, C1-11alkylcarbonylamino, aryl C1-11alkylcarbonylamino, tetrahydrotiryl, morpholinyl, piperazinyl, hydroxypyronyl, xe2x80x94C0-11alkylCOOR1, xe2x80x94C0-11alkylCONR2R3 wherein R1, R2 and R3 are independently selected from hydrogen, C1-C11alkyl, arylC0-C11alkyl, or R2 and R3 are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one C1-C11alkyl, arylC0-C11alkyl substituent.
The term xe2x80x9calkyloxyxe2x80x9d (e.g. methoxy, ethoxy, propyloxy, allyloxy, cyclohexyloxy) represents an alkyl group as defined above having the indicated number of carbon atoms attached through an oxygen bridge. The term xe2x80x9calkyloxyalkylxe2x80x9d represents an alkyloxy group attached through an alkyl group as defined above having the indicated number of carbon atoms.
The term xe2x80x9calkylthioxe2x80x9d (e.g. methylthio, ethylthio, propylthio, cyclohexenylthio and the like) represents an alkyl group as defined above having the indicated number of carbon atoms attached through a sulfur bridge. The term xe2x80x9calkylthioalkylxe2x80x9d represents an alkylthio group attached through an alkyl group as defined above having the indicated number of carbon atoms.
The term xe2x80x9calkylaminoxe2x80x9d (e.g. methylamino, diethylamino, butylamino, N-propyl-N-hexylamino, (2-cyclopentyl)propylamino, hxenylamino, pyrrolidinyl, piperidinyl and the like) represents one or two alkyl groups as defined above having the indicated number of carbon atoms attached through an amine bridge. The two alkyl groups maybe taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 11 carbon atoms with at least one C1-C11alkyl, arylC0-C11alkyl substituent. The term xe2x80x9calkylaminoalkylxe2x80x9d represents an alkylamino group attached through an alkyl group as defined above having the indicated number of carbon atoms.
The term xe2x80x9calkylcarbonylxe2x80x9d (e.g. cyclooctylcarbonyl, pentylcarbonyl, 3-hexenylcarbonyl) represents an alkyl group as defined above having the indicated number of carbon atoms attached through a carbonyl group. The term xe2x80x9calkylcarbonylalkylxe2x80x9d represents an alkylcarbonyl group attached through an alkyl group as defined above having the indicated number of carbon atoms.
The term xe2x80x9calkylcarboxyxe2x80x9d (e.g. heptylcarboxy, cyclopropylcarboxy, 3-pentenylcarboxy) represents an alkylcarbonyl group as defined above wherein the carbonyl is in turn attached through an oxygen. The term xe2x80x9calkylcarboxyalkylxe2x80x9d represents an alkylcarboxy group attached through an alkyl group as defined above having the indicated number of carbon atoms.
The term xe2x80x9calkylcarbonylaminoxe2x80x9d (e.g. hexylcarbonylamino, cyclopentylcarbonyl-aminomethyl, methylcarbonylaminophenyl) represents an alkylcabonyl group as defined above wherein the carbonyl is in turn attached through the nitrogen atom of an amino group. The nitrogen group may itself be substituted with an alkyl or aryl group. The term xe2x80x9calkylcarbonylaminoalkylxe2x80x9d represents an alkylcarbonylamino group attached through an alkyl group as defined above having the indicated number of carbon atoms. The nitrogen group may itself be substituted with an alkyl or aryl group.
The term xe2x80x9carylxe2x80x9d represents an unsubstituted, mono-, di- or trisubstituted monocyclic, polycyclic, biaryl and heterocyclic aromatic groups covalently attached at any ring position capable of forming a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art (e.g., 3-indolyl, 4-imidazolyl). The aryl substituents are independently selected from the group consisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl, C1-11alkyl, arylC1-11alkyl, C0-11alkyloxyC0-11alkyl, arylC0-11alkyloxyC0-11alkyl, C0-11alkylthioC0-11alkyl, arylC0-11alkylthioC0-11alkyl, C0-11alkylaminoC0-11alkyl, arylC0-11alkylaminoC0-11alkyl, di(arylC1-11alkyl)aminoC0-11alkyl, C1-11alkylcarbonylC0-11alkyl, arylC1-11alkylcarbonylC0-11alkyl, C1-11alkylcarboxyC0-11alkyl, arylC1-11lalkylcarboxyC0-11alkyl, C1-11alkylcarbonylaminoC0-11alkyl, arylC1-11alkylcarbonylaminoC0-11alkyl, xe2x80x94C0-11alkylCOOR4, xe2x80x94C0-11alkylCONR5R6 wherein R4, R5 and R6 are independently selected from hydrogen, C1-C11alkyl, arylC0-C11alkyl, or R5 and R6 are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one C1-C11alkyl, arylC0-C11alkyl substituent.
The definition of aryl includes but is not limited to phenyl, biphenyl, naphthyl, dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl, fluorenyl, pyrenyl, thienyl, benzothienyl, isobenzothienyl, 2,3-dihydrobenzothienyl, furyl, pyranyl, benzofuranyl, isobenzofuranyl, 2,3-dihydrdbenzofuranyl, pyrrolyl, indolyl, isoindolyl, indolizinyl, indazolyl, imidazolyl, benzimidazolyl, pyridyl, pyrazinyl, pyradazinyl, pyrimidinyl, triazinyl, quinolyl, isoquinolyl, 4H-quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromanyl, benzodioxolyl, piperonyl, purinyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, benzthiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, oxadiazolyl, thiadiazolyl.
The term xe2x80x9carylalkylxe2x80x9d (e.g. (4-hydroxyphenyl)ethyl, (2-aminonaphthyl)hexenyl, pyridylcyclopentyl) represents an aryl group as defined above attached through an alkyl goup as defined above having the indicated number of carbon atoms.
The term xe2x80x9carylcarbonylxe2x80x9d (e.g. 2-thiophenylcarbonyl, 3-methoxyanthrylcarbonyl, oxazolylcarbonyl) represents an aryl group as defined above attached through a carbonyl group.
The term xe2x80x9carylalkylcarbonylxe2x80x9d (e.g. (2,3-dimethoxyphenyl)propylcarbonyl, (2-chloronaphthyl)pentenylcarbonyl, imidazolylcyclopentylcarbonyl) represents an arylalkyl group as defined above wherein the alkyl group is in turn attached through a carboryl.
The term xe2x80x9csignal transductionxe2x80x9d is a collective term used to define all cellular processes that follow the activation of a given cell or tissue. Examples of signal transduction include but are not in any way limited to cellular events that are induced by polypeptide hormones and growth factors (e.g. insulin, insulin-like growth factors I and II, growth hormone, epidermal growth factor, platelet-derived growth factor), cytokines (e.g. interleukines), extracellular matrix components, and cell-cell interactions.
Phosphotyrosine recognition units/tyrosine phosphate recognition units/phosphotyrosine recognition units are defined as areas or domains of proteins or glycoproteins that have affinity for molecules containing phosphorylatad tyrosine residues (pTyr). Examples of pTyr recognition units include but are not in any way limited to: PTPases, SH2 domains and PTB domains.
PTPases are defined as enzymes with the capacity to dephosphorylate pTyr-containing proteins or glycoproteins. Examples of PTPases include but are not in any way limited to: intracellular PTPases (e.g. PTP-1B, TC-PTP, PTP-1C, PTP-1D, PTP-D1, PTP-D2), receptor-type PTPases (e.g. PTPxcex1, PTPxcex5, PTPxcex2, PTPxcex3, CD45, PTPxcexa, PTPxcexc), dual specificity phosphatases (e.g. VH1, VHR, cdc25) and other PTPases such as LAR, SHP-1, SHP-2, PTP-1H, PTPMEGI, PTP-PEST, PTPxcex6, PTPS31, IA-2 and HePTP and the like.
Modulation of cellular processes is defined as the capacity of compounds of the invention to 1) either increase or decrease ongoing, normal or abnormal, signal transduction, 2) initiate normal signal transduction, and 3) initiate abnormal signal a transduction.
Modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units is defined as the capacity of compounds of the invention to 1) increase or decrease the activity of proteins or glycoproteins with pTyr recognition units (e.g. PTPases, SH2 domains or PTB domains) or to 2) decrease or increase the association of a pTyr-containing molecule with a protein or glycoprotein with pTyr recognition units either via a direct action on the pTyr recognition site or via an indirect mechanism. Examples of modulation of pTyr-mediated signal transduction/modulation of the activity of molecules with pTyr recognition units, which are not intended in any way limiting to the scope of the invention claimed, are: a) inhibition of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; b) inhibition of PTPase activity leading to initiation of normal or abnormal cellular activity; c) stimulation of PTPase activity leading to either increased or decreased signal transduction of ongoing cellular processes; d) stimulation of PTPase activity leading to initiation of normal or abnormal cellular activity; e) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to increase or decrease of ongoing cellular processes; f) inhibition of binding of SH2 domains or PTB domains to proteins or glycoproteins with pTyr leading to initiation of normal or abnormal cellular activity.
A subject is defined as any mammalian species, including humans.
This application relates to compounds having the general structure shown in Formula (A1): 
wherein
(i) Rxe2x80x2 and Rxe2x80x3 are independently selected from the group consisting of hydrogen, halo, cyano, nitro, trihalomethyl, alkyl, arylalkyl,
(ii) Rxe2x80x2xe2x80x3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl
(iii) X is aryl,
(iv) Y is selected from hydrogen or 
wherein (*)indicates a potential point of attachment to X and all other positions are substituted as described below.
(1) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A2): 
wherein at least one of R1, R2 and R3 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2 and R3 are independently selected from the goup consisting of hydrogen, alkyl, substituted alkyl, aryl, arylalkyl.
(2) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A3): 
wherein at least one of R1, R2 and R3 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), ad wherein the remaining of R1, R2 and R3 are independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl, arylalkylcarbonyl.
(3) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A4): 
wherein at least one of R1, R2 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2 is defined as above in Formula (A2).
(4) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A5): 
wherein at least one of R1 and R2 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1 and R2 is defined as above in Formula (A2).
(5) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A6): 
wherein at least one of R1, R2, R3 and R4 substituents has the general structure depicted in Formula(B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2, R3 and R4 have the same definition as R1, R2 and R3 in Formula (A2), with the proviso that when R3 and R4 are selected from substituted phenyl or substituted furyl then the phenyl and furyl substituents exclude hydroxy, halo, trifluoromethyl C1-6alkyl, C1-6alkyloxy, C1-6alkylthio, amino, C1-6alkylamino, di(C1-6alkyl)amino, phenylC1-6alkylamino and di(phenylC1-6alkyl)amino.
(6) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A6): 
wherein R4 is selected from xe2x80x94COR5, xe2x80x94COOR6, xe2x80x94CONR7R8 wherein R5 thru R8 are selected from hydrogen, alkyl, substituted alkyl, aryl, arylalkyl, or R7 and R8 are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at least one alkyl, aryl, arylalkyl substituent, and wherein at least one of R1, R2, and R3 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2 and R3 are defined as above in Formula (A2).
(7) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A6): 
wherein R1, R2, R3 and R4 we defined as above in (6).
(8) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A7): 
wherein R2 is selected from xe2x80x94COR5, xe2x80x94COOR6, xe2x80x94CONR7R8 wherein R5 thru R8 are defined as above in (6) and wherein at least one of R1 and R3 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1 and R3 are defined as above in Formula (A2).
(9) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A8): 
wherein at least one of R1 and R2 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1 and R2 is defined as above in Formula (A2), and wherein m is an integer between 0 and 4 and each R3 is independently selected from the group consisting of halo, nitro, cyano, trihalomethyl, hydroxypyronyl, alkyl, arylalkyl, C0-11alkyloxyC0-11alkyl, arylC0-11alkyloxyC0-11alkyl, C0-11alkylthioC0-11alkyl, arylC0-11alkylthioC0-11alkyl, C0-11alkylaminoC0-11alkyl, arylC0-11alkylaminoC0-11alkyl, di(arylC1-11alkyl)aminoC0-11alkyl, C1-11alkylcarbonylC0-11alkyl, arylC1-11alkylcarbonylC0-11alkyl, C0-11alkylcarboxyC0-11alkyl, arylC0-11alkylcarboxyC0-11alkyl, C1-11alkylcarbonylaminoC0-11alkyl, arylC1-11alkylcarbonylaminoC0-11alkyl, xe2x80x94C0-11alkylCOOR4,xe2x80x94C0-11alkylCONR5R6 wherein R4, R5 and R6 are independently selected from hydrogen, C1-C11alkyl, arylC0-C11alkyl, or R5 and R6 are taken together with the nitrogen to which they are attached forming a cyclic system containing 3 to 8 carbon atoms with at let one C1-C11 alkyl, arylC0-C11 alkyl substituent.
(10) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A8). 
wherein R1 is selected from xe2x80x94COR5, xe2x80x94COOR6, xe2x80x94CONR7R8 wherein R5 thru R8 are defined as above in (6) and wherein R2 has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein m is an integer between 0 and 4 and each R3 is defined as above in (9).
(11) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A9): 
wherein m is an integer between 0 and 3 and wherein R1, R2 each R3 is defined as above in (9).
(12) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A9): 
wherein either R1 or R2 is selected from xe2x80x94COR5, xe2x80x94COOR6, xe2x80x94CONR7R8 wherein R5 thru R8 are defined as in (6) and wherein the remainder of R1 and R2 is defined as above in (9), and wherein m is an integer between 0 and 3 and each R3 is defined as above in (9).
(13) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structure Formula depicted in (A10): 
wherein Z1 and Z2 are independently selected from the group consisting of OR3, SR4, NR5R6 and wherein at least one of R1, R2 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2 is defined as above in Formula (A2), and wherein R3, R4, R5, R6 are independently selected from hydrogen, alkyl, substituted alkyl, alkylcarbonyl, substituted alkylcarbonyl, aryl, arylalkyl, arylcarbonyl, arylalkycarbonyl.
(14) According to the invention, a class of preferred PTPase activity-modulating compounds have the general structural Formula depicted in (A11): 
wherein at least one of R1, R2, and R3 substituents has the general structure depicted in Formula (B) 
wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above in Formula (A1), and wherein the remaining of R1, R2 and R3 are defined as above in Formula (A2).
Preferred compositions of the invention include compositions comprising compounds as defined above in structural formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11) (or pharmaceutically acceptable salts, prodrugs, esters, or solvates of these compounds) in admixture with a pharmaceutically acceptable diluent, adjuvant, or carrier.
Provided according to the invention, therefore, are novel compounds which modulate the activity of PTPase or other molecules with pTyr recognition units) as well as previously known aryl acrylic acid compounds which modulate the activity of PTPase or other molecules with pTyr recognition unit(s).
Another aspect of the present invention provides compositions comprising PTPase modulating compounds of the invention suitable for administration to a mammalian host.
In a preferred embodiment the compounds of the invention act as inhibitors of PTPases, e.g. protein tyrosine phosphatases involved in the regulation of tyrosine kinase signaling pathways. Preferred embodiments include modulation of receptor-tyrosine kinase signaling pathways via interaction with regulatory PTPases, e.g. the signaling pathways of the insulin receptor, the IGF-I receptor and other members of the insulin receptor family, the EGF-receptor family, the platelet-derived growth , factor family, the nerve growth factor receptor family, the hepatocyte growth factor receptor family, the growth hormone receptor family and members of other receptor-type tyrosine kinase families. Further preferred embodiments of the invention is modulation of non-receptor tyrosine kinase signaling through modulation of regulatory PTPases, e.g. modulation of members of the Src kinase family. One type of preferred embodiments of the invention relates to modulation of the activity of PTPases that negatively regulate signal transduction pathways. Another type of preferred embodiments of the inventions relate to modulation of the activity of PTPases that positively regulate signal transduction pathways.
In a preferred embodiment compounds of the inventions act as modulators of the active site of PTPases. In another preferred embodiment the compounds of the invention modulate the activity of PTPases via interaction with structures positioned outside the active sites of the enzymes, preferably SH2 domains. Further preferred embodiments include modulation of signal transduction pathways via binding of the compounds of the invention to SH2 domains or PTB domains of non-PTPase signaling molecules.
Other preferred embodiments include use of the compounds of the invention for modulation of cell-cell interactions as well as cell-matrix interactions.
As a preferred embodiment the compounds of the invention may be used as therapeutics to inhibit PTPases involved in the regulation of the insulin receptor tyrosine kinase signaling pathway in patients with type I diabetes, type II diabetes, impaired glucose tolerance, insulin resistance and obesity. Further preferred embodiments include use of the compounds of the invention for treatment of disorders with general or specific dysfunction of PTPase activity, e.g. proliferative disorders including neoplastic diseases and psoriasis. As another embodiment, the compounds of the invention may be used in pharmaceutical preparations for treatment of osteoporosis.
Preferred embodiments of the invention further include use of compounds of the invention in pharmaceutical preparations to increase the secretion or action of growth hormone and its analogs or somatomedins including IGf-I and IGF-2 by modulating the activity of PTPases or other signal transduction molecules with affinity for phosphotyrosine involved controlling or inducing the action of these hormones or any regulating molecule.
To those skilled in the art, it is well known that the current and potential uses of growth hormone in humans are varied and muti-tudinous. Thus, compounds of the invention can be administered for purposes of stimulating the release of growth hormone from the pituitary or increase its action on target tissues thereby leading to similar effects or uses as growth hormone itself. The uses of growth hormone maybe summarized as follows: stimulation of growth hormone release in the elderly; prevention of catabolic side effects of glucocorticoids; treatment of osteoporosis, stimulation of the immune system; treatment of retardation, acceleration of wound healing; accelerating bone fracture repair; treatment of growth retardation; treating renal failure or insufficiency resulting in growth retardation; treatment of physiological short stature including growth hormone deficient children and short stature associated with chronic illness; treatment of obesity and growth retardation associated with obesity; treating growth retardation assumed with the Pader-Willi syndrome and Turner""s syndrome; accelerating the recovery and reducing hospitalization of burn patients; treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushings syndrome; induction of pulsatile growth hormone release; replacement of growth hormone in stressed patients; treatment of osteochondro-dysplasis, Noonans syndrome, schizophrenia, depressions, Alzheimer""s disease, delayed wound healing and psychosocial deprivation; treatment of pulmonary dysfunction and ventilator dependency; attenuation of protein catabolic responses after major surgery; reducing cachexia and protein loss due to chronic illness such as cancer or AIDS; treatment of hyperinsulinemia including nesidio-blastosis; adjuvant treatment for ovulation induction; stimulation of thymic development and prevention of age related decline or thymic function; treatment of immunosuppresed patients; improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal homeostasis in the frail elderly; stimulation of osteoblasms, bone remodelling and cartilage growth; stimulation of the immune system in companion animals and treatment of disorder of aging in companion animals; growth promotant in livestock and stimulation of wool growth in sheep.
The compounds of the invention may be used in pharmaceutical preparations for treatment of various disorders of the immune system, either as stimulant or suppresor of normal or perturbed immune functions, including autoimmnune reactions. Further embodiments of the invention for treatment of allergic reactions, e.g. asthma, dermal reactions, conjunctivitis.
In another embodiment, compounds of the invention may be used in pharmaceutical preparations for prevention or induction of platelet aggregation
In yet another embodiment, compounds of the invention may be used in pharmaceutical preparations for treatment of infectious disorders. In particular, the compounds of the invention may be used for treatment of infectious disorders caused by Yersinia and other bacteria as well as disorders caused by viruses of other micro-organisms.
Compounds of the invention may additionally be used for treatment or prevention of diseases in animals, including commercially important animals.
Also included in the present invention is a process for isolation of PTPases via affinity purification procedures based on the use of immobilized compounds of the invention using procedures well-known to tose skilled in the art.
The invention is further directed to a method for detecting the presence of PTPases in cell or in a subject comprising
(a) contacting said cell or an extract thereof with labeled compounds of the invention.
(b) detecting the binding of the compounds of the invention or measuring the quantity bound, thereby detecting the presence or measuring the quantity of certain PTPases.
The invention further relates to analysis and identification of the specific functions of certain PTPases by modulating their activity by using compounds of the invention in cellular assay systems or in whole animals.
The invention further provides methods for making compounds (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11) of the present invention having PTPase-modulatory/inhibitory activity. In preferred methods, compounds of the invention are synthesized in a multi-component combinatorial array, which permits rapid synthesis of numerous, structurally related compounds for subsequent evaluation In preferred synthesis protocols, the acrylic acid moiety of a compound is protected during synthesis by, e.g., esterification with a tert-butyl protecting group. Thus, a preferred method of making compounds of the invention comprises use of a protected acrylic acid reagent and removal of the protective group by, e.g., treatment of a precursor ester compound with acid. Optionally, such a method includes further esterifying or salifying the acrylic acid product thereby obtained.
The compounds of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11) may be prepared by procedures known to those skilled in the art from known compounds or readily preparable intermediates. General synthetic procedures and examples are as follow:

Unless otherwise stated, tert-butyl esters were converted to their corresponding carboxylic acids via treatment with a solution of 50% trifluoroacetic acid in dichloromethnne for 1 hour at 23xc2x0 C. The solvent was removed in vacuo and the residue was azeotroped with toluene or acetonitrile to yield the corresponding carboxylic acid.
Method 1
By allowing a compound of formula (1) wherein LG is a suitable leaving group such as bromo, iodo, or triflate to react with compound of formula (2) wherein Z is hydrogen (Heck reaction: J. Org. Chem., 1977, 42, 3903), or trialkyltin (Stille reaction: J. Am. Chem. Soc., 1991, 113, 9585), or B(OH)2 (Suzuki reaction: J. Am. Chem. Soc., 1989, 111, 314) and wherein Rxe2x80x2, Rxe2x80x3, Rxe2x80x2xe2x80x3 and X are defined as above for formula (A1).
These reactions may be carried out neat or in a solvent such as dimethylformamide (DMF), tetahydrofiran (THF), or toluene, in the presence of a catalyst (e.g. Pd(OAc)2, Pd(PPh3)4, Pd2dba3), a ligand (e.g. Ph3P, Ph3As, (o-tolyl)3P) and a base (e.g. K2CO3, CsCO3, Et3N) at temperatures ranging from 23xc2x0 C. to 130xc2x0 C., for 1 to 60 hours.